Thermophilic micro-organisms for ethanol production

ABSTRACT

A thermophilic micro-organism comprising a modification that increases amylase expression and starch hydrolysis compared to wild-type, wherein the modification is insertion of a heterologous amylase gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/673,399, filed Apr. 7, 2010, which is the national stage of PCTApplication No. PCT/GB2008/002782, filed Aug. 12, 2008, the disclosureof each of which is incorporated herein by reference in its entirety,including all figures, tables, nucleic acid sequences, amino acidsequences, and drawings.

FIELD OF THE INVENTION

This invention relates to the production of micro-organisms suitable forthe production of ethanol. In particular, the invention relates to themodification of micro-organisms to enable the utilisation of starch as afermentation substrate.

BACKGROUND TO THE INVENTION

Bacterial metabolism can occur through various different mechanismsdepending on the bacterial species and environmental conditions.Heterotrophic bacteria, which include all pathogens, obtain energy fromoxidation of organic compounds, with carbohydrates (particularlyglucose), lipids and protein being the most commonly oxidised compounds.Biological oxidation of these organic compounds by bacteria results insynthesis of ATP, a chemical energy source. The process also permitsgeneration of more simple organic compounds (precursor molecules) whichare required by the bacterial cell for biosynthetic reactions.

Starch is a naturally abundant carbohydrate and is the principal glucosestorage complex in plants. Starch molecules consist of twopolysaccharides called amylose and amylopectin. Amylose is a linearpolymer of 500 to 20,000 D-glucose subunits, which are linked togethervia a-1,4 glucosidic bonds to form a helical structure. The additionalpresence of a-1,6 glucosidic bonds results in amylopectin, which has abranched structure. Starch generally comprises 20-30% amylose and 70-80%amylopectin. In plant cells, insoluble starch is packed into solidgranules in which amylopectin is clustered in crystalline regions andamylose is distributed throughout. Starch solubility increases withtemperature; amylopectin crystals become gelatinous and granuleseventually dissolve.

Amylase is a calcium-dependant glycoside hydrolase metalloenzyme. Thereare three forms of amylase (α, β and γ) which vary according to thespecific bonds they hydrolyse. Alpha-amylase catalyses the randomhydrolysis of internal a-D-1,4 glucosidic bonds, releasing simplefermentation sugars including glucose, maltose (disaccharides formed bytwo glucose units). Dextrins (short, low molecular weight a-1,4-linkedD-glucose polymers) are released by amylopectin hydrolysis andmaltotriose and maltose are released by amylase hydrolysis. Beta-amylaseacts from the non-reducing end of the starch chain, catalysing thehydrolysis of the second a-1,4 glucosidic bond to cleave two glucoseunits (maltose). Gamma-amylase has the capacity to cleave a-1,6 linkagesin amylopectin. Alpha-amylase is widely synthesized in nature since manyorganisms can digest starch. In human physiology a-amylase is mostprominent in saliva and pancreatic secretions. Microbial a-amylases areclassified as either liquefying (randomly cleaves the polysaccharide toform shorter chains) or saccharifying (produces mono-, di-, ortrisaccharide units), depending on the points of hydrolysis of theglucose polymer chain.

The general process by which bacteria metabolise suitable substrates isglycolysis, which is a sequence of reactions that converts glucose intopyruvate with the generation of ATP. The fate of pyruvate in thegeneration of metabolic energy varies depending on the micro-organismand the environmental conditions. The four principal reactions ofpyruvate are illustrated in FIG. 1.

First, under aerobic conditions, many micro-organisms will generateenergy using the citric acid cycle and the conversion of pyruvate intoacetyl coenzyme A, catalysed by pyruvate dehydrogenase (PDH).

Second, under anaerobic conditions, certain ethanologenic organisms cancarry out alcoholic fermentation by the decarboxylation of pyruvate intoacetaldehyde, catalysed by pyruvate decarboxylase (PDC) and thesubsequent reduction of acetaldehyde into ethanol by NADH, catalysed byalcohol dehydrogenase (ADH).

A third reaction, which also occurs in anaerobic conditions, is theconversion of pyruvate to acetyl CoA, catalysed by pyruvate formatelyase (PFL). Acetyl CoA is subsequently converted into acetaldehyde bythe enzyme acetaldehyde dehydrogenase (AcDH) and ethanol is produced bythe reduction of acetaldehyde catalysed by ADH.

A fourth process is the conversion of pyruvate into lactate which occursthrough catalysis by lactate dehydrogenase (LDH).

There has been much interest in using micro-organisms for the productionof ethanol using either micro-organisms that undergo anaerobicfermentation naturally or through the use of recombinant micro-organismswhich incorporate the pyruvate decarboxylase and alcohol dehydrogenasegenes. The use of such micro-organisms, modified to enhance utilisationof starch as a metabolic substrate, would enable efficient production ofethanol from cheap, abundant, un-refined plant material.

Thermophilic bacteria have been proposed for ethanol production, andtheir use has the advantage that fermentation can be carried out atelevated temperatures which allows the possibility that the ethanolproduced can be removed as vapour at temperatures above 50° C.; thisalso permits fermentation to be carried out using high substrateconcentrations. However, there is a need for improved micro-organismsfor ethanol production from starch-based culture media.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, a thermophilicmicro-organism is modified to increase amylase gene expression comparedto wild-type, wherein a first modification is the insertion of aheterologous amylase gene under the control of a suitable promoter, or aseries of different promoters.

The micro-organism may be further modified to permit increasedproduction of ethanol, via up-regulation of the native pyruvatedehydrogenase gene and inactivation of the native pyruvate formate lyaseand lactate dehydrogenase genes.

The micro-organism of the invention shows increased starch hydrolysisand increased ethanol production compared to wild-type.

According to a second aspect of the present invention, a method for theproduction of ethanol comprises culturing a micro-organism according tothe definition provided above under suitable conditions in the presenceof starch.

DESCRIPTION OF THE DRAWINGS

The present invention is described with reference to the accompanyingfigures, wherein:

FIG. 1 illustrates schematically the metabolic pathway of glycolysis;

FIG. 2 illustrates the pGEM-T Easy® Vector;

FIG. 3 illustrates the hypothetical promoter regions and genes of thePDH complex;

FIG. 4 illustrates the use of pTMO111 for integration of the amylasegene, amyS;

FIG. 5 is the nucleic acid coding sequence of amyS;

FIG. 6 is a graph showing the batch fermentation results for strainTM304 in a 5% w/v soluble starch culture; and

FIG. 7 is a graph showing the batch fermentation results for strainTM333 in a 5% w/v soluble starch culture.

DESCRIPTION OF THE INVENTION

The present invention is based on the modification of a thermophilicmicro-organism to enable enhanced amylase gene expression.

Increasing expression of the amylase gene enables the micro-organism tohydrolyse starch into glucose monomer units which can then be utilisedas glycolytic substrates for the formation of pyruvate and subsequentlyethanol. Methods of increasing amylase expression and enzyme activitypreferably include the use of strong up-stream promoters to regulatetranscription of the gene and incorporation of additional amylase genesthat are expressed at a higher frequency than the native amylase gene.

The thermophilic micro-organism of the invention may be further modifiedto disrupt the expression of the native lactate dehydrogenase gene andto up-regulate the PDH gene.

Inactivating the lactate dehydrogenase gene helps to prevent thebreakdown of pyruvate into lactate, and therefore promotes (underappropriate conditions) the breakdown of pyruvate into ethanol usingpyruvate decarboxylase and alcohol dehydrogenase. It is preferred if thelactate dehydrogenase gene is disrupted by a deletion within or of thegene.

Up-regulating the PDH gene promotes the conversion of pyruvate intoacetyl CoA, which can then be used, under appropriate conditions, toproduce acetaldehyde and eventually ethanol using acetaldehydedehydrogenase. A further advantage of up-regulating PDH is that pyruvatelevels, which have an inhibitory effect on glucose uptake andglycolysis, are reduced. This further promotes ethanol production.

The term “strong promoter” is defined herein as a promoter thatexpresses the corresponding protein to a level greater than 0.5% of thesoluble protein in a cell.

The micro-organism may be any thermophilic micro-organism, but it ispreferred if the micro-organism is of the Bacillus species. Inparticular, it is preferred if the micro-organism is a wild-typemicro-organism of the Geobacillus species, in particular Geobacillusthermoglucosidasius.

In a preferred embodiment, the micro-organisms selected for modificationare said to be “wild-type”, i.e. they are not laboratory-producedmutants. The micro-organisms may be isolated from environmental samplesexpected to contain thermophiles. Isolated wild-type micro-organisms mayhave limited amylase activity, but insufficient to enable ethanolproduction when cultured in a media comprising starch as the main carbonsource. Isolated wild-type micro-organisms will have the ability toproduce ethanol from pyruvate but, unmodified, lactate is likely to bethe major fermentation product.

It is preferable that the micro-organism of the invention has certaindesirable characteristics which permit the micro-organism to be used ina fermentation process. The micro-organism should preferably have norestriction system, thereby avoiding the need for in vivo methylation.It is preferable if the micro-organism is transformable at a highfrequency. Furthermore, the micro-Organism should be stable to at least3% w/v ethanol, preferably at least 5-10% w/v ethanol. Themicro-organism should have a growth rate in continuous culture tosupport dilution rates of 0.3 h⁻¹ and above.

The micro-organism will be a thermophile and will grow in thetemperature range of 40° C.-85° C. Preferably, the micro-organism willgrow within the temperature range 50° C.-70° C. In addition, it isdesirable that the micro-organism grows in conditions of pH 7.2 orbelow, in particular pH 4.5-pH 6.9.

The culture media may preferably comprise at least 1% w/v starch,preferably at least 10% w/v starch, and most preferably at least 20% w/vstarch. The starch may be soluble or insoluble (e.g. grain starch).Other preferred components of the culture media may include, but are notlimited to those listed in Table 1.

TABLE 1 Final Chemical Vol./L Concentration NaH₂PO₄•2H₂O 10 ml 20 mMK₂SO₄ 20 ml 10 mM Citric acid•H₂O 8 ml 8 mM MgSO₄•7H₂O 20 ml 5 mMCaCl₂•2H₂O 4 ml 0.08 mM Sulphate Trace Elements Stock Solution 5 ml SeeTable 2 Na₂MoO₄•2H₂O 0.1 ml 1.65 μM (NH₄)₂SO₄ 12.5 ml 25 μM  1% Biotin*0.3 ml 12 μM 40% Glucose** 3 × 25 ml 3% w/v Antifoam 2.5 ml *Prepared bydissolving 0.1g biotin per 10 ml DMSO **Final glucose concentrationpreferably between 1 and 4% w/vComponents of sulphate trace elements stock solution are shown in Table2.

TABLE 2 Final Medium Chemical gl⁻¹ (ml) Concentration Conc. H₂SO₄ 5.000Zn SO₄•7H₂O 1.440   25 μM Fe SO₄•7H₂O 5.560   100 μM Mn SO₄•H₂O 1.690  50 μM Cu SO₄•5H₂O 0.250    5 μM Co SO₄•7H₂O 0.562   10 μM Ni SO₄•6H₂O0.886 16.85 μM H₃BO₃ 0.080 Deionised H₂O (final vol.) 1000 ml

In a preferred embodiment, a heterologous amylase gene encodes α-amylase(α-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1). It is preferred that theamylase gene is derived from the Geobacillus species, in particularGeobacillus stearothermophilus.

The coding sequence of α-amylase has been elucidated and the techniquesenabling isolation and amplification of the gene are well known in theart. In order to enable the micro-organism of the invention to exhibitincreased amylase expression compared to wild-type, it is preferred thatthe amylase gene is placed under the control of a strong promoter, whichoperates in low-aeration or anaerobic conditions that favour ethanolproduction by thermophilic micro-organisms. The promoter is preferablyan Idh promoter and may be autologous, but is preferably heterologous,and is most preferably derived from the same species as the amylasegene. Examples of suitable promoters include, but are not limited to,P_Idh from G. stearothermophilus NCA1503, P_ferrA from G.stearothermophilus DSM13240 and P_pfl from B. cereus ATCC14579.

In another embodiment of the invention, a series of different strongpromoters are placed upstream of the amylase gene in order to furtherenhance expression. Examples of suitable strong promoters include, butare not limited to, the glyceraldehyde-3-phosphate promoter (P_GAPDH)and amylase promoter from G. stearothermophilus NCA 1503.

The nucleic acid sequence of P_Idh is also known and techniques forcloning and assembling the promoter sequence upstream of the amylasegene are known to the skilled person.

The promoter/amylase sequence can be cloned into a suitable plasmid orexpression vector containing multiple restriction sites. There arenumerous suitable expression vectors which are commercially available,such as the pGEM®-T Easy Vector (FIG. 2). Restriction enzymes can beused to excise the P_Idh/amylase construct as a specific fragment whichcan be ligated into the corresponding restriction site in atemperature-sensitive plasmid such as pUC19 (New England Biolabs). It ispreferable to use a pyruvate formate lysate knock-out plasmid. Theplasmid construct comprising the amylase gene/Idh promoter can then beelectroporated into the micro-organism of the invention and achieveshomologous recombination with genomic DNA. Chromosomal integrants can beselected for on the basis of their resistance to antibacterial agents,such as ampicillin or kanamycin. Amylase activity can also be visualisedas zones of starch clearing, for example on plate assays.

In a preferred embodiment, the micro-organism of the invention isfurther modified by inactivation of the lactate dehydrogenase gene. Thenucleic acid sequence for lactate dehydrogenase is now known. Using thissequence, it is possible for the skilled person to target the lactatedehydrogenase gene to achieve inactivation of the gene through differentmechanisms. It is possible to inactivate the lactate dehydrogenase geneby the insertion of a transposon. However, it is preferred if thelactate dehydrogenase gene is inactivated by the deletion of the genesequence or a portion of the gene sequence. Deletion is preferred asthis avoids the difficulty of reactivation of the gene sequence which isoften experienced when transposon inactivation is used. In a preferredembodiment, the lactate dehydrogenase gene is inactivated by theintegration of a temperature-sensitive plasmid (for example plasmidpUB190-Idh as disclosed in PCT/GB06/01586), which achieves naturalhomologous recombination or integration between the plasmid and themicro-organism's chromosome. Chromosomal integrants can be selected foron the basis of their resistance to antibacterial agents. Theintegration into the lactate dehydrogenase gene may occur by a singlecross-over recombination event or by a double (or more) cross-overrecombination event.

In a further preferred embodiment, the micro-organism is furthermodified to up-regulate PDH. PDH is a large enzyme complex, containingthree units—E1: pyruvate decarboxylase (EC 1.2.4.1, not EC 4.1.1.1), E2:dihydrolipoamide transacetylase, and E3: dihydrolipoamide dehydrogenase.The complex requires several cofactors, including NAD, FAD, coenzyme Alipoic acid and thiamine pyrophosphate (TPP). Four genes code for thecomplex, as the E1 unit is a heterodimer of α and β subunits, and areoften described as pdhA, pdhB, pdhC and pdhD (E1α, E1β, E2 and E3respectively). The E1 unit of PDH requires TPP in the same way that PDC(EC 4.1.1.1) requires TPP and catalyses a similar decarboxylationreaction, but in the presence of coenzyme A and lipoic acid—carried byother enzyme units—the product is acetyl CoA rather than acetaldehyde.However, PDC activity of the E1 unit has been measured when it has notbeen complexed with other units in PDH (Lessard & Perham; The Journal ofBiological Chemistry; 1994, 269:14, 10378-10383; Tomar et al; AppliedMicrobiology and Biotechnology; 2003, 62, 76-82; Frank et al; Science;2004, 306: October 29, 872-876, supplementary data). Accordingly, PDCactivity of EC 1.2.4.1 may be enhanced by the up-regulation of PDH sothat acetaldehyde is produced over and above acetyl CoA. Enhanced PDHactivity is also sought to remove the pyruvate bottleneck observed inLDH inactivated strains to allow more ethanol to be produced with lessacetate and formate as side products.

To this end, the PDH genes and surrounding sequence was isolated usingstandard “genome walking” techniques. Approximately 8.8 kb of DNA wasisolated, sequenced and found to contain the following genes shown inFIG. 3 and Table 3.

TABLE 3 Position Frame (aa's at 5′ Size Gene (bp) Proposed function and3′) (aa) pdf2  746-192 Peptide deformylase −3 (MIT - IER) 184 orf2 868-1497 Unknown- +1 (MQR - IWK) 209 Hypothetical protein pdhA(α)1875-2984 α-subunit of pyruvate +3 (MGA - ESK) 369 hydrogenase pdhA(β)3003-3965 β-subunit of pyruvate +3 (MIQ - INF) 320 dehydrogenase pdhB4058-5368 Dihydrolipoamide +2 (VAP - MEA) 436 transacetylase lpd5373-6785 Lipoamide +3 (MVV - ISK) 470 dehydrogenase orf7 7432-6833Unknown- −1 (MNK - CTE) 199 Hypothetical protein orf8 7964-8647Transposase +2 (MDL - SPP) 227

The hypothetical promoter regions are shown in FIG. 3 (arrows)—oneupstream from the start of pdhA and a possible second promoter ahead ofpdhB. A previous example of a secondary promoter in the PDH cluster wasreported for Bacillus subtilis (Gao et al; Journal of Bacteriology,2002, 184:10, 2780-2788), but most described PDH gene clusters have justone promoter upstream of the cluster (Neveling et al; Biochimica Acta;1998 1385, 367-372. The upregulation can be carried out using techniquesknown in the art. In particular, upregulation can be carried out byintroducing a suitable promoter or enhancer sequence upstream of the PDHcomplex.

The enzyme complex is known to work under both aerobic and anaerobicconditions (Carlsson et al; Infection and Immunity; 1985, 49:3, 674-678)but it is generally considered to be an aerobic enzyme (Ch 15;Principles of Biochemistry; Lehninger, Nelson & Cox; 2^(nd) Ed, WorthPublishers, New York, 1993, p447) with pyruvate formate lyase (PFL) itsanaerobic counterpart. Both enzymes convert pyruvate, formed inglycolysis, to acetyl CoA to feed into the TCA cycle but the cycle onlyworks completely under aerobic conditions. However, as it is desirableto use anaerobic conditions, promoters that operate in anaerobicconditions are preferred for use in the invention. Thus promoters forenzymes believed to work under anaerobic conditions—examples being theLDH promoter (P_Idh from G. stearothermophilus NCA1503), the PFLpromoters (P_pfl from B. cereus ATCC14579, and G. thermoglucosidasiusNCIMB11955) and ferredoxin promoters (P_ferrA from G. stearothermophilusDSM13240)—can be used, as in PCT/GB2007/03699 which is incorporatedherein by reference.

In a preferred embodiment, a further modification is introduced toenhance the PDC activity, thereby promoting the conversion of pyruvateto acetaldehyde. This can be carried out by inactivating E2;dihydrolipoamide transacetylase (EC 2.3.1.12). Inactivation can becarried out in a manner similar to the inactivation of LDH, but with theE2 gene as the target for disruption.

In a further embodiment, a micro-organism of the invention comprises amodification to inactivate the pyruvate formate lyase gene, therebypreventing/reducing the conversion of pyruvate to acetyl CoA andformate. Pyruvate formate lyase (PFL) is the “anaerobic counterpart” topyruvate dehydrogenase (PDH) and converts pyruvate to acetyl CoA andformate (see FIG. 1). While acetyl CoA can be converted to ethanol viaacetaldehyde dehydrogenase (AcHD), formate is an undesired side productwhich has the potential to inhibit growth in ethanolgenic organisms.

PFL was chosen as a target for knockout in order to promote themetabolic flux towards ethanol production and to improve the redoxbalance of the remaining pathway to ethanol synthesis. An additionaladvantage of this work was the elimination of formate production. PFLactivity can be inactivated via transposon insertion, gene deletion orpartial gene deletion to produce a mutant which does not rely onantibiotic selection for the continuation of the altered phenotype. Inthis embodiment, it is preferred that the micro-organism comprises boththe lactate dehydrogenase inactivation and the up-regulation of thepyruvate dehydrogenase, so that, under anaerobic conditions, ethanolproduction is increased.

In a further preferred embodiment, the micro-organism will also compriseheterologous pyruvate decaroboxylase and alcohol dehydrogenase genes.The expression of these heterologous genes results in the production ofenzymes which redirect the metabolism so that ethanol is the primaryfermentation product. The genes may be obtained from micro-organismsthat typically undergo anaerobic fermentation, including Zymomonasspecies, including Zymomonas mobilis.

Methods for the preparation and incorporation of the gene intomicro-organisms are known, for example in Ingram et al, Biotech &BioEng, 1998; 58 (2+3): 204-214 and U.S. Pat. No. 5,916,787, the contentof each being incorporated herein by reference. The gene may beintroduced in a plasmid or integrated into the chromosome, as will beappreciated by the skilled person.

The micro-organisms of the invention are cultured using soluble starchas part of the feedstock. The temperature, pH and other growthconditions can be selected based on known culture requirements.

An embodiment of the present invention will now be described, withreference to the accompanying drawings, in the following example. Twoapproaches for generating integrants of the G. stearothermophilus DSM22α-amylase gene in the genome of G. thermoglucosidasius NCIMB 11955 areoutlined below. The present invention is exemplified, but not limited,by these methods.

EXAMPLE Integration of the Amylase Gene

Approach 1: Fast-Track Strategy Using a NotI Fragment from the ExistingClone, pGEM-LA

Using techniques well known in the art, the α-amylase sequence (amyS)was generated by PCR and joined to the Idh promoter (P_Idh) from 11955.This construct was cloned into the commercially available pGEM-T Easy®expression vector, generating pGEM-LA. As shown in FIG. 2, the ligationsite in the pGEM-T Easy vector is flanked by multiple restriction sites.It is therefore possible to excise the inserted P_Idh/amylase sequenceson a NotI/NotI fragment.

The NotI fragment from pGEM-LA was ligated into the NotI site of the pflknock-out plasmid construct pTMO111 to generate two sibling plasmidconstructs, pTMO139 and pTMO140. These were introduced viaelectroporation into TM242, a stable PFL-negative 11955 strain.Presumptive integrants were selected at 68° C.

The primary transformants (autonomous plasmids) and primary integrantswere tested for amylase production by growth as patches on TGP+0.4%soluble starch plates for 3 days at 60° C., followed by flooding withGram's iodine solution. Large zones of clearing (starch hydrolysis) wereseen around these strains, comparable to a DSM22 control. TM242 gave atrace of activity in this test, much less than DSM22, indicating somebackground activity. Sub-culture of primary integrants in liquid mediain the absence of kanamycin followed by replication of colonies toTGP_kanamycin was used to identify potential double cross-over mutants(DCOs). A total of 32 kanamycin sensitive colonies were tested foramylase activity on TGP+0.4% soluble starch (3 ml in the wells of12-well Costar plates, 3 days at 60° C.). None gave large zones, exceptfor the positive control, DSM22. The majority of strains gave traces ofclearing, similar to the TM242 control, but 5 showed modest yetsignificant zones of clearing. Diagnostic PCR was carried out on genomicpreparations from 2 of these strains, TM319 and TM320, using degenerateprimers bamr66a and bamr72, which were designed based on sequencehomology between known Bacillus PFL sequences. For both strains, asingle PCR product of approximately 3.1 kb was obtained, which ondigestion with NotI gave a double bank of approximately 0.6 kb and aband of approximately 2.0 kb. This is consistent with the expected genereplacement/insertion. The controls gave the expected results,approximately 1.7 kb product from 11955 (wild-type) which did not cutwith NotI and an approximately 1.3 kb product from TM242 which gave adouble bank of approximately 0.6 kb on digestion with NotI. It thereforeappears that these strains (TM319 and TM320) represent integration ofthe amylase construct at the pfl locus.

Approach 2: Generating Amylase Constructs with the P_Idh(NCA) Promoter

The strategy used to place the amylase coding sequence from DSM22 underthe control of P_Idh(NCA) is outlined in FIG. 4. pTMO31 is a plasmidvector comprising ECOR1/SnaB1 pUB110 fragment inserted into the multiplecloning site (mcs) of pUC19 (NEB).

The amylase coding sequence was amplified, via PCR, as an NdeI/NotIfragment, using the pGEM-LA construct as template. P_Idh(NCA) wasamplified as a NdeI/NotI fragment using pTMO49 (produced by cloningP_Idh(NCA 1503) into pTMO23) as a template, and the products were clonedand assembled in pTMO23 (pUC19 with NdeI site removed) to generate theinsertion (gene replacement) constructs pTMO146 and pTMO147 (siblingplasmids). Table 4 details the PCR components for generation ofP_Idh(NCA)/amyS constructs for insertion into 11955.

TABLE 4 PCR fragment Template Primers Size (bp) AmyS cds NdeI/NotIpGEM-LA Bamr87 1657 + primers Bamr88 P_Idh(NCA) NotI/NdeI pTMO49 Bamr89 180 + primers Bamr38

Sequencing of Amylase Clones

The cloned PCR product for the amylase coding sequence generated usingthe bamr87 and bamr88 oligos (see Table 4) was sequenced to checkintegrity. Two independent clones were sequenced. Each showed adifferent single base-pair mutation, which was presumably introduced byPCR. The clone chosen for progression was pTMO135 (PCR product blunt-endligated into the SmaI site of pTMO23). The PCR-induced mutation in thisclone represents a silent mutation, giving the same amino acid andsimilar codon usage, as judged by the published codon usage for G.kaustophilus. The amylase coding sequence from pTMO135 (omitting thestart codon) is shown in FIG. 5. Nucleotides corresponding todiscrepancies between this sequence and the published DSM22 amySsequence (accession number M57457) are underlined. There are 9differences between the pTMO135 sequence (covering 15 bp) and thepublished DSM22 amyS sequence (M57457). The mismatch at 1449 representsthe PCR-induced mutation in pTMO135.

Sequencing of the amylase insert in pTMO139, which has the NotI fragmentfrom pGEM-LA, showed perfect agreement with pTMO135 (except for the1449bp mutation). The amylase coding sequence was then amplified by PCRdirectly from G. stearothermophilus DSM22 genomic DNA (the strain wasobtained from the DSMZ collection). The PCR product obtained (using thebamr87 and 88 oligos as before) was cloned into pTMO23 (to give pTMO145)and sequenced. The sequence was identical to pTMO135 (except for the1449 mutation) and pTMO139, with the exception of bp904. In both pTMO135and pTMO139 bp904 is “A”, but in pTMO145 it is “G”. This introduces anaspartic acid to asparagine mutation at this position. This codon (GACfor aspartic acid) proved to be conserved in all Geobacillus amylasesequences examined, so it is probably a mutation introduced by PCRcloning of the amylase sequence from DSM22. The other discrepancies inthe sequence must be assumed to be errors in the published sequence;alignment with other Geobacillus amylase sequences indicates that thesequence (i.e. in pTMO135 and pTMO145) is far closer to the consensusthan the published sequence (M57457).

Experimental work continued with the pTMO145 and pTMO146 constructs,despite the 904 mutation, as it was clear that the cloned NotI fragmentfrom pGEM-LA did give substantial amylase activity on starch plates.

Generation and Characterisation of DCOs for pTMO145 and pTMO146(P_Idh(NCA)/amyS)

pTMO145 and pTMO146 were introduced into TM242 by electroporation andpresumptive double cross-over (DCO) mutants were selected as outlinedpreviously. A total of 48 presumptive DCOs (kanamycin-sensitive) weretested in plate assays for amylase activity. 18 gave very strong amylaseactivity (the others looked similar to the TM242 control). When patchedto larger sector plates, they gave zones of clearing at least as largeas the DSM22 control. Four of these strains, TM304, TM305, TM311 andTM315, were selected for further work. Genomic DNA was prepared and usedin diagnostic PCR. All four gave results indicating insertion of theamyS gene at the pfl locus. Two amylase mutant strains, TM304 and TM305,were tested for ethanol production in ASYE(0.5%) first with glucose ascarbon source, and then with soluble starch. Testing was conducted inlaw and high aeration conditions. The results are shown in Table 5.TM304 and TM305 produce approximately as much ethanol from solublestarch as from glucose in both high and low aeration models. TM242produces notably lower levels of ethanol on starch compared to glucose,but this effect is considerably less marked in higher aerationconditions. TM304 and TM305 produce similar amounts of ethanol to theTM242 when cultured with glucose, however when the three strains areincubated in the presence of starch it is clear that the superioramylase function of TM304 and TM305 allows the micro-organisms toconvert substantially all of the starch into ethanol. In low aerationconditions this is almost to the same level as with glucose, whilstTM242's ability to produce ethanol from starch is approximately a thirdof its ability to convert glucose to ethanol.

TABLE 5 Strain Carbon source O₂ Glucose Ethanol Acetate Starch* TM304 2%glucose Low 15.9 144.6 3.1 TM305 2% glucose Low 14.3 141.8 3.4 TM242 2%glucose Low 8.7 142.7 3.8 TM304 2% soluble starch Low 3.8 138.4 4.1TM305 2% soluble starch Low 4.6 135.9 3.8 TM242 2% soluble starch Low0.0 52.5 7.6 Yes TM304 2% glucose High 18.2 113.4 19.7 TM305 2% glucoseHigh 10.4 119.8 20.8 TM242 2% glucose High 7.9 127.8 17.3 TM304 2%soluble starch High 16.8 98.9 20.7 TM305 2% soluble starch High 6.7117.9 25.2 TM242 2% soluble starch High 6.5 78.0 23.5 Yes *test forresidual starch; 0.5 ml culture plus 0.2 ml Gram's iodine. All negativeexcept those marked. Inoculum; 100 ul of frozen stock Seed; 10 ml 2TY in50 ml conical, 52° C. O/N, 250 rpm, 1 ml transfer (10%) Production; 10ml ASYE (0.5%) plus C source in 15 or 50 ml falcon, 60° C., 250 rpm, 24hrs

The batch fermentation results for strain TM304 cultured in 5% w/vsoluble starch are shown in FIG. 6. A simple test with Gram's iodineindicated that the DCOs, unlike TM242, hydrolyzed all the starch. Theresults clearly support the utility of the inserted heterologous amylaseunder the P_Idh(NCA) promoter for starch utilization in low aerationconditions.

Generation and Characterisation of DCOs for pTMO150 and pTMO151(P_Idh(NCA)/amyS—no Mutation)

New amyS PCR product from DSM22 genomic DNA was used to generate “clean”DCO constructs, termed pTMO150 and pTMO151. These two plasmids haveP_Idh(NCA) inserted into the pfl gene in opposite orientations. pTMO150is in the same direction of transcription as pfl. pTMO151 is in theopposite direction, as is P_Idh(NCA) in pTMO146 and pTMO147. DCOs weregenerated from both plasmids and two mutants from each were verified byPCR (TM328 and 329 from pTMO150 and TM333 and 335 from pTMO151). Twoamylase mutant strains, TM304 and TM305, were tested. All four mutantstrains, together with TM304, TM305 and TM242 were tested for ethanolproduction in low aeration conditions in ASYE(0.5%), first with 2% w/vglucose as carbon source, and then with 2% w/v soluble starch andfinally 3% w/v soluble starch.

The results, which are shown in Table 6, show that all mutant strainsperformed similarly well in 2% w/v soluble starch, producing over twiceas much ethanol as the parent strain TM242. In 3% w/v starch, TM333produces more ethanol and produces a greater area of clearing on starchplates than the other mutants, indicating that TM333 may be a superiorethanol producer over TM304.

TABLE 6 Strain C source Glucose (mM) EtOH (mM) Starch Area TM328 2%glucose 25.1 128.6 852 TM329 2% glucose 26.7 121.3 871 TM333 2% glucose12.7 135.4 871 TM335 2% glucose 37.5 102.8 860 TM304 2% glucose 3.9149.8 882 TM305 2% glucose 9.5 151.5 886 TM242 2% glucose 20.4 130.8 879TM328 2% starch 5.0 127.5 1817 TM329 2% starch 4.8 132.2 1816 TM333 2%starch 4.8 133.3 1626 TM335 2% starch 12.0 124.8 1836 TM304 2% starch6.7 135.0 1727 TM305 2% starch 6.6 136.1 1716 TM242 2% starch 0.0 62.23803 TM328 3% starch 13.9 139.3 2817 TM329 3% starch 14.1 137.4 2824TM333 3% starch 30.6 151.3 2333 TM335 3% starch 16.9 121.4 2991 TM304 3%starch 31.0 141.2 2440 TM305 3% starch 30.0 140.1 2510 TM242 3% starch0.0 75.6 6306

These results are supported by the results of batch fermentation ofTM333 (carried out under the same conditions as the fermentation ofstrain TM304 (FIG. 6)), which are illustrated in FIG. 7 and suggest thatTM333 is able to utilise all the glucose released from starch.

Comparative testing was carried out to ensure that the mutant strainsTM304 and TM333 performed in a similar fashion to the parent strainTM242 and had not developed any unexpected mutations. The results areshown in Tables 7A and 7B.

TABLE 7A Hrs to Glucose Aeration complete sugar remaining/ Pyruvate/Lactate/ Strain switch consumption OD mM mM mM TM304 3 hr/OD 5.9 6.210.1 0 1 8 TM333 3 hr/OD 5.5 7.4 9.1 0 1 10 TM242 2.5 hr/OD 5.6   7.59.8 0 1 10

TABLE 7B Ethanol Overall yield post ethanol Formate/ Acetate/ Ethanol/anaerobic yield: peak Strain mM mM mM switch g/g ethanol TM304 0 12 3360.41 0.40 TM333 0 16 293 0.44 0.38 TM242 0 13 314 0.44 0.42

1. A thermophilic micro-organism modified by the insertion of aheterologous amylase gene under the control of a suitable promoter, andfurther modified to inactivate the native lactate dehydrogenase gene. 2.The micro-organism according to claim 1, wherein the promoter operatesin low aeration or anaerobic conditions.
 3. The micro-organism accordingto claim 1, wherein the promoter is a heterologous lactate dehydrogenasepromoter.
 4. The micro-organism according to claim 3, wherein thelactate dehydrogenase promoter is derived from Geobacillusstearothermophilus.
 5. The micro-organism according to claim 1, whereinthe amylase gene is under the control of a series of strong promotersincluding a glyceraldehyde-3 phosphate dehydrogenase promoter or anamylase promoter.
 6. The micro-organism according to claim 5, whereinthe promoters are derived from G. stearothermophilus.
 7. Themicro-organism according to claim 1, wherein the lactate dehydrogenasegene, or a portion thereof, has been deleted.
 8. The micro-organismaccording to claim 1, wherein the micro-organism does not comprise aninsertion element in the lactate dehydrogenase gene.
 9. Themicro-organism according to claim 1, wherein a further modificationinactivates the native pyruvate formate lyase gene.
 10. Themicro-organism according to claim 1, further comprising a modificationthat up-regulates pyruvate decarboxylase activity.
 11. Themicro-organism according to claim 1, wherein the dihydrolipoamidetransacetylase gene, or a portion thereof, is deleted.
 12. Themicro-organism according to claim 1, wherein the amylase gene is derivedfrom Geobacillus stearothermophilus.
 13. The micro-organism according toclaim 1, wherein the micro-organism is Geobacillus thermoglucosidasius.14. The micro-organism according to claim 1, wherein the micro-organismdoes not comprise a restriction system.
 15. The micro-organism accordingto claim 1, wherein the micro-organism is stable in a culture mediumcomprising up to 10% w/v ethanol.
 16. The micro-organism according toclaim 1, wherein the micro-organism grows at a temperature from 40°C.-85° C.
 17. A method for the production of ethanol, comprisingculturing a micro-organism under suitable conditions in the presence ofstarch, wherein the micro-organism is a thermophilic micro-organismmodified by the insertion of a heterologous amylase gene under thecontrol of a suitable promoter, and further modified to inactivate thenative lactate dehydrogenase gene.
 18. The method according to claim 17,wherein the culture media comprises at least 10% w/v starch.
 19. Themethod according to claim 17, wherein the method is carried out at atemperature of between 40° C.-70° C., and the micro-organism ismaintained in culture at a pH of between 4 and 7.5.
 20. An animal feed,comprising thermophilic micro-organisms, modified by the insertion of aheterologous amylase gene under the control of a suitable promoter, andfurther modified to inactivate the native lactate dehydrogenase gene.